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New Chemo- and Immunotherapeutic Approaches in Chagas’ Disease

New Chemo- and Immunotherapeutic Approaches in Chagas’ Disease. Rosa A. Maldonado Medina Federal University of São Paulo (UNIFESP). Chagas’ Disease (American trypanosomiasis). Chagas’ disease affects 16-18 million people in Latin America, from Mexico to Argentina.

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New Chemo- and Immunotherapeutic Approaches in Chagas’ Disease

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  1. New Chemo- and Immunotherapeutic Approaches in Chagas’ Disease Rosa A. Maldonado Medina Federal University of São Paulo (UNIFESP)

  2. Chagas’ Disease (American trypanosomiasis) • Chagas’ disease affects 16-18 million people in Latin America, from Mexico to Argentina. • The ethiological agent is the protozoan parasite • Trypanosoma cruzi • Current treatment (benznidazole, Rochagan) is partially effective (~60% cure). • Recently, drug-resistant strains have been found. .

  3. Carbohydrates Lipids Proteins Chemotherapeutic Target Properties a) The molecule must perform essential functions in the parasite. Basic Metabolism Life or death b) It can be selectively inhibited: Present in T. cruzi, but not in mammals

  4. Oleate (D-12) desaturase (ODTc ): A New Chemotherapeutic Target in Chagas’ Disease

  5. X* = CoA or ACP (acyl carrier protein) or phospholipid. • OD is absent in humans, then it can be selectively inhibited. • Linoleic acid is a major component of GPI anchor of T. cruzi trypomastigote mucins and phospholipids. So, the product of OD seems to performessential functions for the parasite. OD fulfills all the requirements as a potential chemotherapeutic target.

  6. P1, P2= degenerated primers P1 gDNA PCR P2 (1003 bp) High similarity with D12-desaturase from plants Sac I Genomic clone Sac I 3 kb Mannosyl Transferase Size selection library 5´-ODTc 3´- missing Hind II Sac I Sph I Kpn I Kb 3´-RACE PCR Sph I Sph I ~8 Kb 3.0 Sac Sac I I Sac Sac I I Sac Sac I I Mannosyl Transferase I OD II OD Tc Tc 2.7 Kb 2.7 Kb 3 Kb 3 Kb 1.2 Kb 1.2 Kb Cloning of the T. cruzi Oleate Desaturase (ODTc)

  7. Dm28c S.X10/7 0.45 Chromosomal localization Y CL CL-Brener Mb Mb Mb 1.03 0.95 0.85 0.85 0.45 Strain Chrom.Mb Dm28c SylvioCL YCL Band X10/7Brener IX 1.03 VII 0.95 VI 0.85 I 0.45 Phylogenetic groups: I (sylvatic cycle): Dm28c, Sylvio X10/7 II (domestic cycle): Y, CL Hybrid: CL Brener

  8. Alignment of TrypanosomatidODs vs. PlantOD T.cruzi L.major T.brucei B.officinalis T.cruzi L.major T.brucei B.officinalis T.cruzi L.major T.brucei B.officinalis T.cruzi L.major T.brucei B.officinalis T.cruzi L.major T.brucei B.officinalis T.cruzi L.major T.brucei B.officinalis T.cruzi L.major T.brucei B.officinalis Putative transmembrane motifs His-rich motifs: possibly participating in catalysis PSORT II - k-nearest neighbor (k-NN) algorithm:44.4 %: endoplasmic reticulum

  9. ODTc Xba I Xba I Homologous expression of ODTc fused to GFP 5`-Xba I gDNA Xba I -5` PCR GFP ODTc 1 x 106 epimastigotes/ 200mL Eco RV Xba I Xba I Neor Electroporation pTEX 50 mg plasmid AMPr Selection LIT G418 (100 mg/mL) 1% blood Transfected parasites

  10. Co-localization of ODtc in the Endoplasmic Reticulum OD-GFP ER-Tracker Blue-White DPX Phase Merge

  11. Southern - gDNA Southern - cDNA Cf Hm Ls Tf Tr Tl Hm Bc Cf La Ls Kb Kb 10.0 5.0 3.0 2.5 2.5 1.5 1.5 1.0 1.0 Chromosomal localization M Tc Cf Hm TconTf Tc Cf Hm Tcon Tf M Tc Es Ls Tc Es Ls Mb Mb 1.1 0.95 0.82 0.95 0.75 0.82 0.68 0.61 0.61 0.45 0.45 0.38 Molecular characterization of OD in other trypanosomatids

  12. Biochemical studies of the native OD activityin different trypanosomatids

  13. T. cruzi Epimastigote T. cruzi Trypomastigote Cf Cf La Hm Ls La Hm Ls TAG TAG TAG TAG C18:1 C18:1 C18:1 Erg DAG Erg DAG Erg DAG PA/MAG PE/PME PA/MAG PI PE/PME IPC PE/PME PE/PME PI LPI PI/IPC PC PC PC LPC LPC LPC Metabolic labeling and lipid analysis 1010 parasites + 5mCi [14C]-oleic acid 16 h, at 28 or 37ºC Washed Freeze-dried Lipid extraction (C:M, 2:1; C:M:W, 1:2:0.8) Total lipids HPTLC Autoradiography

  14. Fatty acid hydrogenation of Trypanosomatid phospholipids Phospholipids (PC, PE, PI) Alkaline hydrolysis Methylation +/-Hydrogenation RP-HPTLC Autoradiography C18:3 C18:3 C18:2 C18:2 C18:1 C18:1 C18:0 C18:0 Hydrogenation + - + - + - + - + - + - Tc epi Tc tryp Cf La Hm Ls

  15. CONCLUSIONS • ODTcgene has 2 copies in tandem array and is transcribedin all T. cruzi stages. • The enzymeis localized in the endoplasmic reticulum. • The enzymes -12 and -15 desaturases are active in trypanosomatids. • OD seems to be promising chemotherapeutic target in Chagas’ disease, leishmaniasis and African trypanosomiasis, as well as in fungal infections.

  16. The Spider Antimicrobial Peptide Gomesin as a New Adjuvant in the Immunotherapy against T. cruzi

  17. Antimicrobial peptides (AMP) • Involved in innate immunity; found from bacteria to mammals • Cationic molecules composed of 12 to 50 amino acids • Frequently rich in cysteine residues • Positively charged in physiological pH, and with amphypathic structure in -helix, -sheet or both • Primary action mechanism is the permeabilization of the microbial membrane

  18. Gomesin • Isolated from hemocytes of the Brazilian tarantula spider Acanthoscurria gomesiana (Silva Jr. et al, JBC, 2000) • Mass of 2270 Da, 18 amino acids (ZCRRLCYKQRCVTYCRGRa), two disulfide bonds (Cys6-11 e Cys2-15) • Antibacterial (Gram-negative and Gram-positive) and antifungal activity

  19. Gomesin kills T. cruzi epimastigotes in vitro DL50 = 6.5 mM Survival (%) Concentration (mM)

  20. Control Liposomal Gomesin Free Gomesin Empty Liposome In vivo activity of gomesin in the experimental infection of mice BALB/c by T. cruzi 1.5×107 1.0×107 Parasitemia (No. of parasites/mL) 0.5×107 0 0 1 2 3 4 5 6 7 8 9 10 Time (days after infection)

  21. 100 Control Control Liposomal Gomesin 80 Free Gomesin 60 Survival (%) Empty Liposome 40 Non Infected + Free Gomesin 20 Non Infected + Liposomal Gomesin 0 0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 Time (days after infection) In vivo activity of gomesin in the experimental infection of mice BALB/c by T. cruzi

  22. How gomesin was protecting the animals if it was not by directly killing the parasites? • One possibility was that gomesin like defensins (Biragyn et al., Science, 2002) was acting on the mouse innate immune system, inducing pro-inflammatory cytokines, through the activation of Toll-like receptors. • TLRs are primary sensors of the innate immunity. They recognize Pathogen-Associated Molecular Patterns (PAMPs)(e.g. LPS, GPIs, etc), initiating signaling pathways that stimulate the host defense against microorganisms.

  23. METHODOLOGY • Transfected CHO/CD14 cells: CHO/CD14/TLR2 • CHO/CD14/TLR4 • CHO/CD14/mMD2 • CHO/CD14/mTLR4 • Gomesin stock solution was cleaned-up of endotoxins using an affinity column of polimyxin B (depletion of LPS) • The stimulation of the TLR was measured by the expression of CD25 on the surface of the cells by FACS mutants not able to signal via TLR4

  24. CD25 expression Fold increase 12 0.12 0.001 1.2x10-6 Gomesin concentration (nM) 10.0 CHO/CD14/mMD2 CHO/CD14/mTLR4 7.5 CHO/CD14/TLR2 CD25 expression Fold increase CHO/CD14/TLR4 5.0 2.5 0.0 None Gom S.a. LPS Stimulus Activation of TLR4 by Gomesin Gomesin activates TLR4 at low nanomolar to picomolar range

  25. TLR-4 3 GOMESIN 2 MD-2 CD14 ? Plasma membrane MyD88 Cytoplasm IRAK Signaling cascade 4 IkB 6 NF-kB 5 P Synthesis of Pro-inflammatory Cytokines and Chemokines Activation of immune response genes Nucleus Gomesin activates Toll-like receptor 4

  26. CONCLUSIONS • Gomesin stimulates TLR4, so it is a new PAMP. • Gomesin could be used as an adjuvant of the innate immune response against Chagas’ infection and, probably, other infectious diseases.

  27. ACKNOWLEDGMENTS • University of São Paulo (USP) • ICB • Lab. Parasite Glycobiology • Igor C. Almeida • Renata K. Kuniyoshi • Lab. of Biochem. and Immunol. of Arthropodes • Sirlei Daffre • Marcello R. Burgierman • IQ • Maria Terêsa Miranda • Iolanda Cuccovia • Federal University of São Paulo (UNIFESP) • Antônio Miranda • Marcos Fazio University of Dundee Alan Fairlamb Financial support FAPESP/ CNPq WHO/TDR The Wellcome Trust-UK

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